Apolipoprotein B (also known as ApoB, apolipoprotein B-100; ApoB-100, apolipoprotein B-48; ApoB-48 and Ag(x) antigen), is a large glycoprotein that serves an indispensable role in the assembly and secretion of lipids and in the transport and receptor-mediated uptake and delivery of distinct classes of lipoproteins. Apolipoprotein B was cloned (Law et al., PNAS USA 82:8340-8344 (1985)) and mapped to chromosome 2p23-2p24 in 1986 (Deeb et al., PNAS USA 83, 419-422 (1986)). ApoB has a variety of functions, from the absorption and processing of dietary lipids to the regulation of circulating lipoprotein levels (Davidson and Shelness, Annu. Rev. Nutr., 20:169-193 (2000)). Two forms of ApoB have been characterized: ApoB-100 and ApoB-48. ApoB-100 is the major protein component of LDL, contains the domain required for interaction of this lipoprotein species with the LDL receptor, and participates in the transport and delivery of endogenous plasma cholesterol (Davidson and Shelness, 2000, supra). ApoB-48 circulates in association with chylomicrons and chylomicron remnants which are cleared by the LDL-receptor-related protein (Davidson and Shelness, 2000, supra). ApoB-48 plays a role in the delivery of dietary lipid from the small intestine to the liver.
Susceptibility to atherosclerosis is highly correlated with the ambient concentration of apolipoprotein B-containing lipoproteins (Davidson and Shelness, 2000, supra). Elevated plasma levels of the ApoB-100-containing lipoprotein Lp(a) are associated with increased risk for atherosclerosis and its manifestations, which may include hypercholesterolemia (Seed et al., N. Engl. J. Med. 322:1494-1499 (1990), myocardial infarction (Sandkamp et al., Clin. Chem. 36:20-23 (1990), and thrombosis (Nowak-Gottl et al., Pediatrics, 99:E11 (1997)).
Apolipoprotein B knockout mice (bearing disruptions of both ApoB-100 and ApoB-48) have been generated which are protected from developing hypercholesterolemia when fed a high-fat diet (Farese et al., PNAS USA. 92:1774-1778 (1995) and Kim and Young, J. Lipid Res., 39:703-723 (1998)). The incidence of atherosclerosis has been investigated in mice expressing exclusively ApoB-100 or ApoB-48 and susceptibility to atherosclerosis was found to be dependent on total cholesterol levels.
In view of such findings, significant efforts have been made to modulate serum cholesterol levels by modulating ApoB expression using therapeutic nucleic acids, e.g., antisense oligonucleotides, ribozymes, etc. (see, e.g., U.S. Pat. No. 7,407,943, which is directed to modulation of ApoB using antisense oligonucleotides). More recent efforts have focused on the use of interfering RNA molecules, such as siRNA and miRNA, to modulate ApoB (see, Zimmermann et al., Nature, 441: 111-114 (2006), U.S. Patent Publication Nos. 20060134189 and 20060105976, and PCT Publication No. WO 04/091515). Interfering RNA molecules can down-regulate intracellular levels of specific proteins, such as ApoB, through a process termed RNA interference (RNAi). Following introduction of interfering RNA into the cell cytoplasm, these double-stranded RNA constructs can bind to a protein termed RISC. The sense strand of the interfering RNA is displaced from the RISC complex, providing a template within RISC that can recognize and bind mRNA with a complementary sequence to that of the bound interfering RNA. Having bound the complementary mRNA, the RISC complex cleaves the mRNA and releases the cleaved strands. RNAi can provide down-regulation of specific proteins, such as ApoB, by targeting specific destruction of the corresponding mRNA that encodes for protein synthesis.
Despite the high therapeutic potential of RNAi, two problems currently faced by interfering RNA constructs are, first, their susceptibility to nuclease digestion in plasma and, second, their limited ability to gain access to the intracellular compartment where they can bind RISC when administered systemically as free interfering RNA molecules. These double-stranded constructs can be stabilized by the incorporation of chemically modified nucleotide linkers within the molecule, e.g., phosphothioate groups. However, such chemically modified linkers provide only limited protection from nuclease digestion and may decrease the activity of the construct.
In an attempt to improve efficacy, investigators have employed various lipid-based carrier systems to deliver chemically modified or unmodified therapeutic nucleic acids, including anionic (conventional) liposomes, pH sensitive liposomes, immunoliposomes, fusogenic liposomes, and cationic lipid/nucleic acid aggregates. In particular, one lipid-based carrier system, i.e., the stable nucleic-acid lipid particle (SNALP) system, has been found to be particularly effective for delivering interfering RNA (see, U.S. Patent Publication No. 20050064595 and U.S. Patent Publication No. 20060008910 (collectively referred to as “MacLachlan et al.”)). MacLachlan et al. have demonstrated that interfering RNA, such as siRNA, can be effectively systemically administered using nucleic acid-lipid particles containing a cationic lipid, and that these nucleic acid-lipid particles provide improved down-regulation of target proteins in mammals including non-human primates (see, Zimmermann et al., Nature, 441: 111-114 (2006)).
Even in spite of this progress, there remains a need in the art for improved SNALPs that are useful for delivering therapeutic nucleic acids, such as siRNA and miRNA, to the liver of a mammal (e.g., a human), and that result in increased silencing of target genes of interest in the liver, such as ApoB. Preferably, these compositions would encapsulate nucleic acids with high-efficiency, have high drug:lipid ratios, protect the encapsulated nucleic acid from degradation and clearance in serum, be suitable for systemic delivery, and provide intracellular delivery of the encapsulated nucleic acid. In addition, these nucleic acid-lipid particles should be well-tolerated and provide an adequate therapeutic index, such that patient treatment at an effective dose of the nucleic acid is not associated with significant toxicity and/or risk to the patient. The present invention provides such compositions, methods of making the compositions, and methods of using the compositions to introduce nucleic acids, such as siRNA and miRNA, into the liver, including for the treatment of diseases, such as hypercholesterolemia (e.g., atherosclerosis, angina pectoris or high blood pressure).